Relationships between structure and function have been investigated in three flavoproteins each of which contains an active center cystine: lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase. The amino acid residue sequence around the active center cystine has been rigorously conserved through the long evolution between E. coli and pig in lipoamide dehydrogenase, thus between a prokaryote enzyme and its mitochondrial, eukaryotic successor. Similar studies in yeast and E. coli glutathione reductase are in progress. Characterization of the mechanism of E. coli glutathione reductase has been completed and comparison with the yeast enzyme has been shown to be qualitative rather than quantitative, as was the case with E. coli and pig heart lipoamide dehydrogenase. Determination of the sequences around each of the cysteines in the two lipoamide dehydrogenases is in progress. The positions of the thiol-containing peptides in a tryptic digest of the pig heart enzyme have been determined in peptide maps. Thiols involved in the formation of disulfide upon treatment with copper have been tentatively identified. The thiol protected from alkylation by NAD has also been tentatively identified. The catalytic and structural properties of the two lipoamide dehydrogenases are being studied under conditions of progressive and reversible denaturation. Studies on the enzyme isolated from human heart are anticipated.